Analysis of action potentials, transients, and ion flux in excitable cells
Inventors
Cerignoli, Fabio • Gehalot, Piyush • McDonough, Patrick M. • Price, Jeffrey H. • Whittaker, Ross J.
Assignees
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Publication Number
US-10928308-B2
Publication Date
2021-02-23
Expiration Date
Abstract
Video recordings from two or more optical channels are produced, processed, and analyzed simultaneously in order to provide quantitative analysis of action potentials, calcium transients and ionic flux in excitable cells loaded with voltage or ion sensitive dyes with distinct excitation and emission wavelengths. The specific wavelengths of fluorescent light emitted from each dye are separated and recorded. The recordings are mutually registered and cytometric analysis is performed to provide a quantitative analysis of the action potentials, calcium transient, and/or ionic flux on a cell-by-cell and well-by-well basis in microtiter plates. The cells are then fixed, labeled for other biomarkers, and scanned again. The resulting fixed cell images are registered with the live cell recordings and analyzed; missing cells that were washed off are detected relative to the live recordings, and cytometry data from live and fixed cell scans is collated cell-by-cell.
Core Innovation
The invention is a method executable by an instrument for producing simultaneous video recordings of a magnified field of view from multiple optical channels. The recordings are made from a sample containing cells loaded with two or more fluorescent dyes designed to respond to action potentials, calcium transients, ion concentrations, or a flow of ions across a cell membrane, and the instrument is configured with an optical path to support simultaneous multi-channel imaging corresponding to two or more different intracellular components.
The instrument includes a motorized stage for positioning a region of the sample above the objective and an autofocus module that moves the microscope objective to focus by collecting a stack of images at different focus positions. The autofocus module calculates a degree of focus or a sharpness index, derives a best focus position, and sets focus to the best focus position, with the stack collected as different fluorescent colors for corresponding live cell channels or as images collected on the same fluorescent channel as a corresponding live cell channel.
An incubation chamber contains the sample and maintains temperature, carbon dioxide, and oxygen at preset levels under control of a control system that records two or more videos simultaneously at varying levels of light. The quantitative processing and analysis include correcting for mirroring, X-Y shift, rotation, and magnification differences between channels from the same field of view, segmenting cells into cell masks, and generating measurements from the cell peripheries and cell masks of changes in intensity over time of the fluorescent dyes.
The invention further encompasses a post-fixation branch in which the sample is fixed and rescanned, with images recorded for the label colors in fixed cells. The method includes registration of fixed with live recordings, detection of cells lost during fixation, and collation of cytometry datasets such that cytometric measurements from live cells and fixed cells are combined for comparison of corresponding labels.
Claims Coverage
The independent claim covers a multi-channel, simultaneous video-recording method with instrument autofocus, controlled incubation, channel-to-channel digital registration, cell segmentation and periphery-based intensity kinetics extraction, and quantitative characterization of action potentials, calcium transients, and ion/ion-flow dynamics. Dependent claims further refine the method by adding electrical stimulation in multiwell plate wells, oxygen control, subcellular region masks, fixed-sample rescanning, fixed/live registration, detection of lost cells, and collation of live and fixed cytometry measurements into a single dataset.
Simultaneous multi-channel video recordings of fluorescent dye-loaded cells for action potentials, calcium transients, and ion behavior
A method executable by an instrument producing simultaneous video recordings of a magnified field of view from multiple optical channels for a sample containing cells loaded with two or more fluorescent dyes responding to action potentials, calcium transients, ion concentrations, or a flow of ions across a cell membrane.
Autofocus by collecting an image stack and setting a best focus position for channel handling
An autofocus module moves the microscope objective relative to the sample by collecting a stack of images at different focus positions, calculates a degree of focus or sharpness index, derives a best focus position, and sets focus to the best focus position, with images collected as different fluorescent colors for corresponding live cell channels or on the same fluorescent channel as a corresponding live cell channel.
Controlled incubation chamber conditions for temperature, carbon dioxide, and oxygen
An incubation chamber contains the sample and maintains temperature, carbon dioxide, and oxygen at preset levels.
Simultaneous dual/multi-video recording and quantitative kinetic analysis by channel registration, cell masking, and intensity-over-time extraction
A control system records two or more videos simultaneously to record varying levels of light corresponding to two or more different intracellular components; recordings are processed by correcting mirroring, X-Y shift, rotation, and magnification differences between channels from the same field of view, segmenting videos into cell masks, generating measurements from cell peripheries and cell masks of changes in intensity over time of fluorescent dyes, plotting changes in intensity versus time, and extracting measurements that characterize shape and duration of cellular action potentials, calcium transient, ion concentrations, and/or ion flow.
Multiwell electrical stimulation via a stimulator arm with electrodes
The method further includes a stimulator arm that automatically positions a pair of electrodes into wells of a multiwell plate to apply a user-defined electrical stimulation protocol to cells.
Subcellular region segmentation and subcellular/periphery intensity kinetics
The method segments cell videos and labels subcellular regions using multiple optical channels, then generates measurements of changes in fluorescence intensity over time from the cell periphery and subcellular region masks.
Post-fixation second scan with recording label color images in fixed cells
The method scans the sample a second time and records images of the label colors in fixed cells.
Fixed-to-live image registration with detection of washed-off or missing cells
The method includes registration of fixed with live recordings and detection of cells lost during fixation.
Collation of live and fixed cytometry measurements into a single dataset
The method combines cytometric measurements from live cells and fixed cells into a single dataset to study and compare corresponding labels.
Overall, the claim set covers simultaneous multi-channel kinetic image cytometry with autofocus-driven channel handling under controlled incubation, digital registration across channels, cell and subcellular masking for intensity-over-time quantification, and extraction of action potential, calcium transient, and ion/ion-flow dynamic parameters. Dependent claims extend the workflow to electrical stimulation, oxygen control, and fixed-sample rescanning with fixed/live registration, missing-cell detection, and collation of live and fixed cytometry measurements.
Stated Advantages
Provides quantitative measurements including one or more of cellular action potentials, calcium transients, ion concentrations, and/or flow ions across a membrane.
Enables plotting changes in intensity of fluorescent dyes versus time and extracting measurements characterizing shape and duration of cellular action potentials, calcium transients, ion concentrations, and/or ion flow.
Corrects mirroring, X-Y shift, rotation, and magnification differences between video recordings of different channels from the same field of view.
Supports segmentation into cell masks and generates periphery and subcellular region measurements of changes in intensity over time.
Enables comparison of corresponding labels by combining cytometric measurements from live cells and fixed cells into a single dataset.
Documented Applications
Studying and comparing corresponding labels by combining cytometric measurements from live cells and fixed cells into a single dataset.
Quantitative kinetic image cytometry for excitable-cell dynamics, including cellular action potentials, calcium transients, and ion/ion-flow across a membrane.
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